As described in the Introduction to this SCOR application, it is now recognized that estrogen directly affects the vasculature in two principal ways: estrogen causes rapid vasodilation, and estrogen activates gene expression and longer-term changes in blood vessel cells. Both the rapid and the longer-term effects of estrogen are thought to be important to the cardiovascular protective effects of the hormone. Project 4 of this SCOR is based on extensive unpublished preliminary data and explores a specific hypothesis related directly to the overall SCOR hypothesis: estrogen receptors mediate both rapid (non-genomic) activation and longer-term (genomic) expression of nitric oxide synthase (NOS) enzymes, affording protection against ischemic cardiovascular diseases and their sequelae. In Specific Aim 1, the mechanism of rapid (non- genomic) endothelial NOS activation by estrogen is explored, based on new data demonstrating that eNOS activation by estrogen in endothelial cells is mediated by an entirely novel function of the estrogen receptor ERalpha, a protein thought until now to function solely as a transcription factor. Signaling events mediating the rapid activation of eNOS by ERalpha are explored in endothelial and COS cell studies of (a) structure- function of ERalpha domains and residues mediating rapid activation of eNOS by estrogen; (b) ERalpha-mediated activation of eNOS via the MAP kinase pathway; and (c) Involvement of Src kinase in mediating ERalpha stimulation of eNOS. In Specific Aim 2, the longer-term, genomic actions of estrogen to protect against vascular injury are explored by studies of estrogen-mediated increases in vascular iNOS gene expression. These studies are based on extensive new data that strongly support that ERbeta induction of VSMC iNOS is critical to estrogen's vascular protective effects. This hypothesis is tested in Specific Aim 2 with studies of (a) ERbeta regulation of iNOS gene expression in cultured VSMC from WT, ERalpha KO and ERbeta KO mice; (b) vasomotion in endothelium-denuded mouse vascular rings from WT, ERalphaKO, ERbetaKO and iNOS KO mice; and (c) Estrogen effects on the vascular injury response to iNOS KO mice. By examining the role of estrogen receptors in both the rapid, non-genomic and longer-term, genomic effects of estrogen on vascular eNOS and iNOS, respectively, Project 4 reflects the overall hypothesis for this SCOR proposal and directly complements studies proposed in SCOR Projects 1, 2, 3 and 5.